1. Field of the Invention
This invention relates to a method for modulating the specific activity of a purified protein product while purifying the protein. More particularly, the specific activity of a purified protein is modulated by addition of a controller protein at one or more points during the course of a purification protocol.
2. Description of Related Art
Interferon is a protein which is produced by a host organism upon infection by viruses, or by an immunoreaction such as an antigen-antibody reaction. Interferon has an antiviral affect.
There are several known processes used to produce interferon. Leukocytes or lymphoblastoid cells can be stimulated to produce interferon with appropriate viruses or lectins. Fibroblast cells may also be stimulated to produce interferon with viruses or a synthetic nucleic acid. Recent developments in recombinant DNA technology make it possible to produce interferon from cellular culture, i.e., from either mammalian cells or micro-organisms such as bacteria or yeast which are transformed with a plasmid carrying an interferon gene.
Interferon is classified into three categories according to its origins. Leukocytes and lymphoblastoid cells stimulated with viruses produce interferon-alpha, those cells stimulated with lectins produce interferon-gamma and fibroblast cells stimulated with a synthetic nucleic acid produce interferon-beta.
When interferon is harvested from cell culture, it normally is found in a crude supernatant liquid which must be further purified in order to obtain a product which is suitable for clinical use. Numerous processes or protocols have been developed to effect such purification. These processes usually involve several complex steps wherein the final interferon yield may be anywhere between 10% to 70% of the starting material. The majority of human interferon used today is produced and purified essentially according to the procedure developed by Kari Cantell et al. as described in "Partial Purification of Human Leukocyte Interferon On a Large Scale" appearing in Methods in Enzymology, Vol. 78, page 499-505, the contents of which are incorporated herein by reference. Techniques which have been employed to purify interferon include precipitation, gel filtration, ion exchange chromatography, gel electrophoresis, affinity chromatography, and high pressure liquid chromatography. These techniques take advantage of various physical and chemical properties associated with the protein.
U.S. Pat. No. 4,503,035 to Pestka, et al., describes a process for purifying proteins, particularly proteins having molecular weights in excess of 12,000 kilodaltons involving applications of high performance liquid chromatography to provide homogenous interferon having a specific activity of from 0.9.times.10.sup.8 to 4.times.10.sup.8 units/mg. of protein when assayed on the MDBK bovine cell line and from 2.times.10.sup.6 -7.6.times.10.sup.8 units/mg. of protein when assayed on the AG 1732 human cell line. Specific activity for interferons and other proteins is defined as the number of units of biological activity per milligram of protein mass. Alpha interferon preparations made according to the Cantell method normally run between 1.0-10.0.times.10.sup.6 units per milligram of protein.
In addition to specific activity, other criteria for assessing the relative purity of a purified protein include molecular weight determination, mass spectroscopy, and sequencing. Specific activity is useful in assessing the relative purity of the protein product and is consequently useful in determining effective dosage of the protein. Thus, the ability to effectively control the specific activity of the end product of the purification process is a desirable goal.